The present invention generally relates to a method of preparing a eukaryotic viral vector.
Gene transfer to eukaryotic cells has many potential uses, both in vivo and in vitro. Gene transfer can be used, for example, to treat an animal prophylactically and/or therapeutically. Gene transfer also can be used for research purposes, such as, for example, to investigate the cellular functions of particular genes and to identify the molecular basis of specific diseases. Recombinant eukaryotic viral vectors have become a preferred method of gene transfer for many researchers and clinicians. These vectors allow for efficient and effective transfer of genes to eukaryotic cells and offer a safe method for treating animals (e.g., humans) in a clinical setting.
While researchers and clinicians have enjoyed the many advantages of eukaryotic viral vectors for gene transfer to eukaryotic cells, the difficulty of constructing stocks of these viral vectors has impeded the rate at which new and useful gene transfer experiments and protocols have been developed. Because of their large size, recombinant eukaryotic viral vectors typically are produced within a host bacterial cell via homologous recombination. The host bacterial cell typically is transfected with two or more expression vectors (e.g., plasmids) containing homologous nucleotide regions, and a double homologous recombination event occurs. Following the homologous recombination event, additional processing steps are typically required in order for the expression vector (e.g., plasmid) containing the recombinant eukaryotic vector to be actively expressed in a eukaryotic cell. These intermediate processing steps include, for example, purification of the expression vector from the bacterial cell and linearization of the vector using a restriction enzyme.
These intermediate processing steps can be both costly and time-consuming. Accordingly, there remains a need for improved methods of generating recombinant eukaryotic viral vectors which minimize, or even eliminate, the need for intermediate processing steps. The present invention seeks to provide such a method. These and other advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
The invention provides a method of producing a eukaryotic viral vector. The method comprises contacting a eukaryotic cell, which comprises a unique enzyme that nicks or cleaves a DNA molecule, with a recombinant phage vector. Alternatively, the method comprises contacting a eukaryotic cell, which does not comprise a unique enzyme that nicks or cleaves a DNA molecule, simultaneously or sequentially, in either order, with (i) a unique enzyme that nicks or cleaves a DNA molecule and (ii) a recombinant phage vector. The recombinant phage vector comprises the DNA molecule comprising (a) a eukaryotic viral vector genome comprising a coding sequence, (b) a phage packaging site that is not contained within the eukaryotic viral vector genome, and (c) a promoter that is operably linked to the coding sequence. The DNA molecule enters the eukaryotic cell, and the unique enzyme nicks or cleaves the DNA molecule in the eukaryotic cell in at least one region not contained within the eukaryotic viral vector genome, whereupon a eukaryotic viral vector is produced.